Interruption of KLF5 acetylation promotes PTEN-deficient prostate cancer progression by reprogramming cancer-associated fibroblasts

Inactivation of phosphatase and tensin homolog (PTEN) is prevalent in human prostate cancer and causes high-grade adenocarcinoma with a long latency. Cancer-associated fibroblasts (CAFs) play a pivotal role in tumor progression, but it remains elusive whether and how PTEN-deficient prostate cancers reprogram CAFs to overcome the barriers for tumor progression. Here, we report that PTEN deficiency induced Krüppel-like factor 5 (KLF5) acetylation and that interruption of KLF5 acetylation orchestrated intricate interactions between cancer cells and CAFs that enhance FGF receptor 1 (FGFR1) signaling and promote tumor growth. Deacetylated KLF5 promoted tumor cells to secrete TNF-α, which stimulated inflammatory CAFs to release FGF9. CX3CR1 inhibition blocked FGFR1 activation triggered by FGF9 and sensitized PTEN-deficient prostate cancer to the AKT inhibitor capivasertib. This study reveals the role of KLF5 acetylation in reprogramming CAFs and provides a rationale for combined therapies using inhibitors of AKT and CX3CR1.


II. Supplemental Tables
Supplemental Table 1.Pathological features of p-AKT+ prostate cancer samples.

Supplemental Figure 2 .
Functional annotations of differential gene expression caused by the interruption of Klf5 acetylation in Pten-deficient prostate tumor cells.(A, B) Top 20 significant (adjusted p-value < 0.05) Gene Ontology (GO) Biological Process sets in anterior prostates (AP, A) and dorsal prostates (DP, B). (C) Central network plot (CNET plot) for the most significantly enriched GO Biological Process sets and their genes (adjusted p-value < 0.05) associated with deacetylated Klf5 in AP. (D) The differentially expressed genes caused by Klf5 KR knockin were closely associated with the altered genes caused by SMAD4 knockout (Nature, 2011), as indicated by GSEA.Supplemental Figure 3. Single cell transcriptomic assay clusters different cell types in Pten-deficient mouse prostates.(A) The distribution of 10 cell clusters in four mouse prostates, as visualized by UMAP.(B) Dot plots of the top 10 marker genes of each cluster in the scRNA-seq of mouse prostates (n = 61713 cells).The annotation of each cluster is shown in Fig. 4. (C) Representative markers of luminal cells are shown as violin plots.(D) Infercnv analysis of prostate epithelial cells suggests that the most significant genome variants occur in Krt4+ luminal cluster.(E, F) Cellchat analysis reveals differential strength of interaction (E) and top differential microenvironmental signaling (F) caused by deacetylation of Klf5.Supplemental Figure 4. Fgf9 in fibroblasts is induced by Klf5 deacetylation in Pten-deficient prostate tumor cells and serves as a functional ligand of FGFR1.(A) Dot plots of the Fgf expression in different cell clusters of mouse prostates, as indicated by scRNA-seq.Cluster numbers refer to the annotations in Fig. 4A.(B) The mRNA levels of Fgfs were determined by RNA-seq in mouse prostates with indicated genotypes.Only genes with Fpkm > 1, at least in one group, are shown.+/+, PB Cre ;Pten -/-;Klf5 WT/WT ; +/KR, PB Cre ;Pten -/-;Klf5 WT/KR ; KR/KR, PB Cre ;Pten -/-;Klf5 KR/KR .Data are shown in mean ± S.E.M. (C, D) FGF9 induced the activation of FGFR1 signaling, as indicated by the Western blotting assay detecting p-ERK Thr202/Tyr204 and p-FRS2 Y436 .DU 145 cells were treated as indicated in a dose curve (C) and a time curve (D).(E) Silence of FGFR1 suppressed the activation of ERK and FRS2, as indicated by Western blotting.G5 and G6 are two different FGFR1 shRNAs.DU 145 cells were treated with FGF9 (25 ng/mL) as indicated time.Supplemental Figure 5. Klf5 deacetylation enhances TNF signaling in prostate tumor microenvironment.(A) TNF signaling activities of each cell cluster were calculated by Gene Set Variation Analysis (GSVA) and plotted as a heatmap.TNF signaling related datasets were collected from MSigDB.(B) Fibroblasts in PB Cre ;Pten - /-;Klf5 KR/KR (KR) group receives enhanced TNF signaling from Krt4+ luminal, macrophages and neutrophils relative to PB Cre ;Pten -/-;Klf5 W/W (WT) group, as indicated by Cellchat.(C) TNF-α was enhanced by Klf5 KR knockin in both epithelial cells and CD11b+ macrophages, as indicated by IF staining.Scale bars, 50 μm.White arrowheads indicate CD11b+/TNF-α+ cells.White arrows indicate CD11b-/TNF-α+ cells.White stars indicate nuclear staining of TNF-α, which was excluded from the statistical analysis of TNF-α+ cells on the right.Supplemental Figure 6.Klf5 deacetylation enhances signaling crosstalk between prostate cancer cells and iCAFs.(A) Dot plots of top 10 marker genes of Krt4+ luminal and different fibroblast clusters in scRNA-seq (n = 35343 cells).(B) KR knockin enhances the strength of interaction between Krt4+ luminal cells and iCAFs.(C) Dot plots of Fgfs in Krt4+ luminal and different fibroblast clusters.(D) Top differential signaling crosstalk between Krt4+ luminal and fibroblasts caused by KR knockin.Cellchat was used to generate panels B and D. Supplemental Figure 7. CX3CR1 induced by deacetylated Klf5 is a potential activator of FGFR1 signaling.(A) A heatmap of FGFR1 activities induced by top differential ligands of Krt4+ luminal autocrine signaling.NicheNet was used to calculate top differential ligands between KR and WT group in scRNA-seq.GSVA was used to evaluate the FGFR1 activities.(B, C) Consistent differentially expressed genes in anterior prostates (AP) and dorsal prostates (DP).The selected genes are with FDR < 0.05 in one lobe and p value < 0.01 in the other lobe.Their gene expression levels are shown in a heatmap (B) and their reported functions are summarized in a table (C).Supplemental Figure 8. CX3CR1 inhibitor suppresses organoid formation and sensitizes PDX to AKT inhibitors.(A) CX3CR1 inhibitor AZD8797 (50 nM) selectively suppresses the organoid formation of mouse prostate cancer cells with deAc-KLF5 in the context of Pten deficiency.Two-tailed Student's t-tests were performed.ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.(B, C) Treatment of Pten knockout GEMM by capivasertib suppressed TGF-β signaling, reduced Ac-Klf5, and enhanced Frs2 phosphorylation, as indicated by IHC staining and Western blotting.(D) PTEN-deficient PDXs on NSG mice were treated by AKT inhibitor capivasertib and/or CX3CR1 inhibitor JMS-17-2, as indicated in the figures daily.The tumor growth of PDX is shown as tumor volume curve for each PDX on NSG mice.(E) Body weight of NSG mice during therapy.(F) The expression levels of Ac-KLF5 were evaluated by quantitative analysis of mean staining intensities (MSI).Supplemental Figure 9. Correlation of FGF9 and CX3CR1 with FGFR1 activation in prostate cancer samples from TCGA database.(A-C) Correlation of FGF9 and FGFR1 activation.(D-F) Correlation of CX3CR1 and FGFR1 activation.Single-sample geneset enrichment assay (ssGSEA) was used to identify the FGFR1 activation for 499 cancer samples by using three different REACTOME genesets.The gene expression levels of FGF9 and CX3CR1 were normalized into a z-score.Pearson analyses were performed in panels A-F.

Table 2 .
Clinical parameters of patient samples in tissue microarray PRC1021.Adenocarcinoma samples with intact IHC staining are using in this study and their pathological data are listed.

Table 3 .
Primers used for realtime qPCR.

Table 4 .
Antibodies used in this study.